1. Field of the Invention
This invention relates to bleomycin products and the method of making and using same, and more specifically, it relates to the advantageous use of fluorescent-labelled bleomycin analogs as probes for identifying cells which are resistant or sensitive to bleomycin or bleomycin derivatives and further relates to the use of such analogs as therapeutic agents for treating malignant cells responding to bleomycin or bleomycin derivative treatment.
2. Description of the Prior Art
Antineoplastic agents are those which inhibit or prevent the growth and spread of neoplasms or malignant cells. The antineoplastic agent bleomycin (BLM) refers to a group of peptides with antitumor activity widely used in the treatment of various cancers, such as squamous cell carcinoma, testicular carcinoma and Hodgkin's disease. See, Lazo, J. S., et al., "Malignant Cell Resistance to Bleomycin Group Antibiotics," Anticancer Drug Resistance, pp. 267-280, (CRC Press, 1989); Carter, S. K., "Bleomycin: More Than a Decade Later," Bleomycin Chemotherapy, pp. 3-35, (Academic Press, Inc., 1985).
The cellular determinants responsible for regulating the toxicity of BLM remain unclear. However, the primary target for the therapeutic action of the BLM class of compounds is thought to be nuclear DNA. See Umezawa, H., "Advances in Bleomycin Studies,", Bleomycin: Chemical, Biochemical and Biological Aspects, pp. 24-36, (Springer-Verlag, 1979); Lazo, J. S., et al., "Initial Single-strand DNA Damage and Cellular Pharmacokinetics of BLM A.sub.2," Biochem. Pharmacol., 38: 2207-2213, (2989). It is believed, for example, that bleomycin causes strand scission and fragmentation of DNA and may, to a lesser extent, inhibit synthesis of RNA. Nonetheless, the magnitude of DNA cleavage and cytotoxicity do not always correlate. See Berry, D. E., et al., "DNA Damage and,Growth Inhibition in Cultured Human Cells by Bleomycin Congeners," Biochemistry, 24: 3207-3213, (1986); Smith, P. T. "Ferrous-iron Mediated Enhancement of DNA Damage and Recovery Potential in Bleomycin-treated Human Cells," Biochem. Pharmacol., 36: 475-480, (1987).
There is also some evidence to suggest that nonnuclear lesions may also be responsible for cell death seen with BLM. For example, BLM-Fe complexes can produce lipid peroxidation in vitro and in vivo. See Lazo, J. S., et al., "Anticancer Drug Resistance, supra; Ciriolo, M. R. , et al., "A Comparative Study of the Interactions of Bleomycin with Nuclei and Purified DNA," J. Biol. Chem., 264: 1443-1449, (1989). These complexes can also produce rapid perturbations of the plasma membrane fluidity in cultured cells. See Bailly, C., et al., "Plasma Membrane Perturbations of KB3 Cells Induced by the Bleomycin-iron Complex," Cancer Res., 50: 385-392, (1990). Thus, it is possible that BLM affects the plasma membrane or other organelles. See Vyskocil, F., et al., "Bleomycin Stimulates Both Membrane (Na+--K+) ATPase and Electrogenic (Na+--K+) Pump and Partially Removes the Inhibition by Vanadium Ions," Biochem. Biophys. Res. Comm., 116: 783-788, (1983); Sun, I. L., et al., "Bleomycin Control of Transplasma Membrane Redox Activity and Proton Movement in HeLa Cells," Biochem. Pharmacol., 34: 617-619, (1985).
In addition to the uncertain nature of the mechanisms whereby BLM affects cells, little is known about cellular BLM uptake or the dynamics of intracellular BLM distribution, because only limited amounts of the drug appear to enter cells and current analytical methods to monitor intracellular BLM are not sufficiently sensitive. Furthermore, certain cells appear to be BLM sensitive, while others are BLM resistant, and the reasons for this are not always clear, nor is it always possible to gauge the relative degrees to which certain cells are BLM-resistant or sensitive.
To understand the mode(s) of action of BLM cytotoxicity, it is important to elucidate (a) the mechanisms by which BLM enters the cell, and (b) the cytoplasmic fate of BLM following internalization. Additionally, since alteration in BLM uptake is one mechanism by which cells become resistant to BLM, understanding the process of internalization as well as of intracellular trafficking of BLM could help further clarify the cellular basis of BLM resistance.
One means of analyzing a drug's intracellular fate is to take advantage of its intrinsic fluorescent. The intrinsic fluorescent of drugs has, in the past, been useful in defining the cellular pharmacology of anticancer agents. For example, several investigators have studied the mechanisms of cytotoxicity and cellular resistance of the anticancer agent, adriamycin, by taking advantage of its intrinsic fluorescent. See Willingham, M. C., et al., "Single Cell Analysis of Daunomycin Uptake and Efflux in Multidrug-resistant and Sensitive KB Cells: Effects of Verapamil and Other Drugs," Cancer Res., 46: 5941-5946, (1986); Lane, P., et al., "Temperature Dependence Studies of Adriamycin Uptake and Cytotoxicity," Cancer Res., 47: 4038-4042, (1987); Herweijer, H., et al., "A Rapid and Sensitive Flow Cytometric Method for the Detection of Multidrug-Resistant Cells," Cytometry, 10: 463-468, (1989).
BLM has intrinsic fluorescent but, unfortunately, studies using this property of BLM have not been informative because the intrinsic fluorescent intensity of BLM is too low to be of practical utility as a cellular probe. Detailed studies of internalization as well as cellular accumulation and localization of BLM and BLM-like compounds have also been severely limited because of difficulty in obtaining higher specific activity [.sup.3 H]BLM.
Another approach to investigating a drug's intracellular activity is to use fluorescent analogs of the drug. Fluorescent analogs of peptides, hormones and drugs have proved valuable alternatives to using radiolabelled species for studying the processes of cellular internalization and intracellular trafficking. See Wang, Y. L., et al., "Methods in Cell Biology," Vol. 29. San Diego, Calif.: Academic Press, Inc., (1989). Fluorescein has been conjugated to methotrexate to identify transport deficient phenotypes. See Guadray, P., et al., "Fluorescent Methotrexate Labeling and Flow Cytometric Analysis of Cells Containing Low Levels of Dihydrofolate Reductase," J Biol. Chem., 261: 6285-6292, (1986); Assaraf, Y. G., et al., "Identification of Methotrexate Transport Deficiency in Mammalian Cells using Fluoresceinated Methotrexate and Flow Cytometry," Proc. Natl. Acad. Sci. (USA), 84: 7154-7158, (1987); Assaraf, Y. G., et al., "Characterization by Flow Cytometry of Fluorescein-methotrexate Transport in Chinese Hamster Ovary Cells," Cytometry, 10: 50-55, (1989).
The cellular uptake and targets of estramustine have been probed using a dansylated derivative. See Sterns, M. E., et al., "Dansylated Estramustine, a Fluorescent Probe for Studies of Estramustine Uptake and Identification of Intracellular Targets," Proc. Natl. Acad. Sci. , (USA), 82:8483-8487, (1985).
Fluorescein isothiocyanate (FITC) has been successfully conjugated to various pharmacological agents such as methotrexate and testosterone. See Gapski, G. R., et al., "Synthesis of a Fluorescent Derivative of Amethopterin," J. Med. Chem., 18: 526-528, (1975); Evarian, C., et al., "The Preparation of Three Fluorescent-labelled Derivatives of Testosterone," Steroids, 35: 610-619, (1980).
However, fluorescent-labelled bleomycin and bleomycin derivatives have not, to the inventors' knowledge, heretofore been identified. Accordingly, there is a need in the art to synthesize a new BLM analog that possesses enhanced fluorescent properties and which enables characterizing the biological properties of BLM and BLM analogs in vitro and in vivo, as well as in cultured BLM-sensitive and BLM-resistant cells.